CN113755506B - A kind of gene related to regulation of lateral branch differentiation of tea tree source and its encoded product and application - Google Patents

Abstract

A gene related to the differentiation regulation of tea tree source collateral and a coding product and application thereof belong to the technical field of biology. The invention provides a gene CsGH3.1 related to the regulation and control of tea plant source collateral differentiation and a coding product thereof, and provides application of the gene. The research shows that CsGH3.1 is closely related to the lateral branch differentiation of tea trees, and the expression product of the gene can increase the number of secondary branches, shorten the length of the secondary branches and reduce the tillering sites of an arabidopsis over-expression plant. The invention can be widely applied to crop breeding, especially tea tree breeding.

Description

A kind of gene related to regulation of lateral branch differentiation of tea tree source and its coding product and application

technical field

The invention belongs to the field of biotechnology, and in particular relates to a gene related to regulation and control of side branch differentiation of tea tree origin, its coding product and application.

Background technique

Lateral branch differentiation is a major determinant of plant morphogenesis and one of the important agronomic traits affecting crop yield and quality. Tea tree (Camellia sinensis L.) is an important foliar economic crop in my country, and its axillary bud germination and lateral branch growth are closely related to the yield and quality of tea. The top dominance of the tea tree is very obvious and the differentiation nodes of the side branches are relatively high. In terms of production, it is necessary to artificially regulate the growth of the side branches to promote the formation of a large number of new shoots of the tea tree. Therefore, it is an urgent need for tea production to discover the genes related to the regulation of tea branch development and to breed tea varieties with lower branch positions and reasonable branch numbers. On the other hand, the completion of tea tree genome sequencing and the increasing improvement of related technologies such as genetics and functional genomics, especially the breakthroughs in the field of plant growth and development regulation, have provided powerful theories and technologies for accelerating the cultivation of tea tree varieties. support.

Auxin is a key hormone that regulates plant growth and development. The key role of auxin concentration on axillary bud development has been clarified, and the balance of auxin in plants must be strictly controlled. The auxin in plants mainly exists in two forms: free state and bound state. Among them, the free state IAA is the main form of active auxin in plants, while the bound state auxin is temporarily formed by the combination of indole acetic acid and other substances into conjugates. Lost biological activity.

In the regulatory mechanism of auxin homeostasis, the most extensively studied auxin early response genes (Primary-response genes). IAA amide synthetase (IAA-amido synthetase, Gretchen Hagen 3, GH3) family protein is a kind of auxin early response protein that widely exists in plants, which can maintain the growth of plants by catalyzing the chelation reaction between excess IAA and amino acids Steady state. So far, genome-wide identification and functional studies of the GH3 gene family have been reported in at least 19 plant species including Arabidopsis thaliana, rice, soybean, tomato, apple, cotton, and kiwifruit. There are 20 GH3 genes in the Arabidopsis genome, which can be divided into three subfamilies (subfamily I, II and III) according to the structure and function of GH3 protein, and subfamily II has IAA amino acid binding enzyme activity. The plant GH3 gene is extensively involved in the regulation of various plant growth and development processes, which has been confirmed and received extensive attention. For example, overexpression of the GH3 gene causes a decrease in the level of endogenous free IAA, resulting in unique low-auuxin plant phenotypes, including plant dwarfing, early tillering, and increased tillering.

Contents of the invention

Aiming at the differences existing in the prior art, the purpose of the present invention is to provide a technical solution for genes related to the regulation of lateral branch differentiation of tea tree origin, their encoded products and their applications.

The present invention is specifically realized through the following technical solutions:

One aspect of the present invention provides a gene CsGH3.1 related to the regulation of tea tree-derived collateral differentiation, the DNA sequence of which is shown in SEQ ID No.1.

Another aspect of the present invention provides a protein encoded by a gene CsGH3.1 related to the regulation of tea tree-derived collateral differentiation, including any of the following proteins:

1) A protein having an amino acid sequence as shown in SEQ ID No.2;

2) Substituting or deleting the amino acid sequence shown in SEQ ID No.2 by one or more amino acid residues;

3) A derivative protein with the amino acid shown in SEQ ID No. 2 added and having the same activity as the amino acid residue sequence shown in SEQ ID No. 2.

Further, the present invention provides an application of a gene CsGH3.1 related to regulation of lateral branch differentiation of tea tree origin in cultivating transgenic plants.

Further, the present invention provides an application of a gene CsGH3.1 related to regulation of lateral branch differentiation of tea tree origin in crop breeding.

Furthermore, the present invention provides an application of a gene CsGH3.1 related to the regulation of lateral branch differentiation of tea tree origin in regulating the homeostasis of plant endogenous auxin.

Further, the present invention provides an application of a gene CsGH3.1 related to regulation of lateral branch differentiation of tea tree origin in regulating plant lateral branch differentiation.

Further, the present invention provides an application of a gene CsGH3.1 related to the regulation of lateral branch differentiation of tea tree origin in increasing the number of secondary branches of plants and reducing the position of tiller nodes.

The beneficial effect of the present invention is, utilize RT-PCR technique, clone obtains CsGH3.1 gene; Utilize fluorescence quantitative PCR (QPCR) to clarify the expression situation of CsGH3.1 gene in the tea tree axillary bud growth and development process induced by pruning and its relationship with Correlation of auxin (including bound auxin and free auxin IAA) content; Transgenic Arabidopsis plants with overexpression of CsGH3.1 gene were obtained by Agrobacterium transformation method, and further revealed that CsGH3.1 gene is in Arabidopsis important role in the regulation of lateral branch differentiation in mustard. The segregation and cloning of the gene plays a very important role in promoting the cultivation of tea tree varieties with ideal branching phenotype.

Description of drawings

Figure 1 shows the tea tree axillary bud phenotype after 16 days of pruning (a), the expression profile of CsGH3.1 during the development of tea tree axillary buds induced by pruning (b) and the content changes of auxin (including bound auxin and free auxin IAA) (c).

Fig. 2 is an electrophoresis image of the ORF full-length amplification product of the CsGH3.1 gene. Lane 1-2 is the 1799bp CsGH3.1 gene fragment.

Fig. 3 is a schematic diagram of pCambia2301 vector transformed by overexpression of CsGH3.1 gene. Note: Endonuclease sites are underlined.

Figure 4 is the homozygous identification of CsGH3.1 overexpressed Arabidopsis lines. (a) PCR identification of overexpression lines; (b) GUS activity identification of overexpression lines.

Figure 5 is a phenotype comparison between Arabidopsis wild-type and overexpression lines. (a) Phenotypes of 4-week-old wild-type (WT) Arabidopsis and CsGH3.1 overexpression lines; (b) 10-week-old CsGH3.1 overexpression Arabidopsis and wild-type phenotypes; (c) wild-type Flower and apical pod growth of Arabidopsis and CsGH3.1 overexpression lines.

Fig. 6 is the phenotype statistics of collateral development in wild-type (WT) Arabidopsis and CsGH3.1 overexpression lines. Note: PB (primary branches) represents primary side branches; SB (secondary branches) represents secondary side branches; T (Tillers) represents tillers.

Fig. 7 is the tissue differential expression pattern of tea tree CsGH3.1 gene in Arabidopsis overexpressed plants (a); the expression pattern of 8 Arabidopsis AtGH3 subfamily II genes in Arabidopsis leaves (b).

Detailed ways

The present invention utilizes RT-PCR technology to obtain the ORF full-length sequence of CsGH3.1 gene; utilizes fluorescent quantitative PCR (QPCR) technology to confirm that CsGH3.1 gene may participate in the regulation of endogenous auxin homeostasis and thus regulate the development of tea tree branches ;Arabidopsis transgenic plants with overexpression of CsGH3.1 were obtained by Agrobacterium transformation method; statistical results showed that overexpression of CsGH3.1 could increase the number of secondary branches of Arabidopsis thaliana, reduce tiller nodes and reduce secondary branching branch length. In summary, the isolation, cloning and biological function analysis of CsGH3.1 gene will play an important role in promoting the breeding of crop varieties with ideal plant type, especially the breeding of tea tree varieties with ideal plant type.

Realize the concrete technical steps of the present invention as follows:

1) Analysis of expression characteristics of CsGH3.1 gene. The axillary buds at the first node below the incision at seven time points from 0 hours to 16 days after pruning in summer, and the axillary buds at the corresponding nodes of the control tea tree (not pruned) were taken respectively. The total RNA was extracted and reverse transcribed to obtain cDNA; the induced expression of CsGH3.1 gene was determined by QPCR technology and tea tree CsGAPDH as an internal reference gene.

2) Functional study of CsGH3.1 regulating the growth and development of tea tree axillary buds induced by summer pruning. Determination of endogenous auxin content: select the axillary bud samples of 0h, 12h, 24h, 48h, 4d, 8d, and 16d after pruning, and measure the endogenous free auxin IAA and amide-bound auxin (IAA-amido, including IAA-Asp and IAA-Ala) content.

3) Isolation and cloning of CsGH3.1 gene and the acquisition of CsGH3.1 overexpression Arabidopsis transgenic lines. ①Using RT-PCR technology, the full-length ORF sequence of CsGH3.1 gene was obtained; ②The full-length ORF of CsGH3.1 was inserted into the 35S promoter of pCambia2301, and the overexpressed CsGH3.1 gene was obtained by Agrobacterium transformation Transgenic lines of Arabidopsis thaliana were further identified by GUS staining and PCR, and two homozygous lines of the T ₃ generation, CsGH3.1-OX-Line1 and CsGH3.1-OX-Line4, were finally screened.

4) Functional study of CsGH3.1 regulating the branching phenotype of CsGH3.1 overexpressed Arabidopsis lines. ①Statistics of the number, length and tiller node position of primary and secondary branches of Arabidopsis wild-type and CsGH3.1 overexpression lines respectively; ②Determination of Arabidopsis wild-type and CsGH3.1 overexpression lines respectively Expression of tea tree CsGH3.1 gene in different tissues (including roots, stems, leaves, flowers and siliques); determination of eight Arabidopsis thaliana AtGH3 subfamilies in leaves of Arabidopsis wild-type and CsGH3.1 overexpression lines Expression of II members, including AtGH3.1.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention.

Example 1: Research on the regulatory role of CsGH3.1 in the growth and development of tea tree axillary buds induced by summer pruning 1) Summer pruning treatment

Pruning is an important cultivation measure in the tea tree production process. At present, the three-dimensional canopy cultivation tea garden that only picks spring tea and does not pick summer and autumn tea generally adopts secondary pruning to cultivate the canopy. The first time is to carry out heavy pruning after the end of spring tea; the second time is to carry out another pruning at around July 20, raising 20cm from the original pruning height, and then let it grow. Collect the axillary buds at the first node below the cut after pruning in the summer of 2019, take samples at different time points 0h, 12h, 24h, 48h, 4d, 8d, and 16d, immediately immerse them in liquid nitrogen, and store them at -80°C for later use; The uncut tea trees were kept as the control group, and the same sampling operation was carried out with the samples of the experimental group.

2) Phenotypic changes of tea tree axillary buds induced by pruning and the expression characteristics of CsGH3.1

①Phenotype analysis

The results of phenotype analysis showed that compared with the control group, the phenotype of the axillary buds at the first node below the incision changed significantly after 16 days of pruning. The volume of leaf buds induced by pruning was much larger than that of the control group, and the leaf buds grew significantly and were more fat, indicating that pruning significantly induced the development of tea tree leaf buds (Fig. 1a).

②QPCR analysis

The methods of RNA extraction and cDNA synthesis were the same as above. SsoFast ^TM probes supermix kit (BIO-RAD) was used for QPCR, and the tea tree CsGAPDH gene was used as an internal reference gene, and detected by CFX96 ^TM Real-Time system (BIO-RAD) fluorescent quantitative PCR instrument; the reaction system and procedures refer to the product manual. The primers and information are as follows:

GH3.1-F1: 5'-ATGTGATTGTAACGGGTGC-3' (as shown in SEQ ID No.3)

GH3.1-R1: 5'-GAGTTGGGTTCGTGAGGGA-3' (as shown in SEQ ID No.4)

GAPDH-F: 5'-TTGGCATCGTTGAGGGTCT-3' (as shown in SEQ ID No.5)

GAPDH-R: 5'-CAGTGGGAACACGGAAAGC-3' (shown in SEQ ID No. 6).

As shown in Figure 1b, the expression level of CsGH3.1 gene in axillary buds was significantly up-regulated within 12-48 hours after pruning, and returned to the 0-hour level 4 days after pruning.

3) Changes of auxin content in tea tree axillary buds induced by pruning

In order to further study the regulatory mechanism of CsGH3.1 in the growth and development of tea tree axillary buds induced by pruning, the changes in the content of endogenous auxins (free IAA and amide-bound compounds of IAA) were detected, and the relationship between CsGH3.1 expression and endogenous auxin was analyzed. Correlation of source auxin content. A total of 8 IAA amido compounds were detected in this assay, but only 2 were detected, namely IAA-Asp and IAA-Ala. The results showed that the content of each amide compound changed dynamically with the content of free IAA respectively, which was highly synchronous with the change of IAA content. For example, within 0-24h and 48h-4d after pruning, the contents of IAA-Asp and free IAA both increased; within 24-48h and 4-8d, the content of free IAA decreased or increased slowly. The overall content decreased accordingly. The overall change trend of the content of the two amide compounds is also similar (Figure 1c), 12h and 24h are significantly higher than 0h, 48h decreased to the level of 0h, and then increased sharply. The changes of IAA-Ala and IAA-Asp contents were both synchronous and alternate, and could accumulate rapidly in a short period of time, and the content of IAA-Ala reached the peak at 12h. In addition, the content of bound compounds mainly fluctuated greatly within 4 days, while the content of free IAA continued to increase after pruning for 8 days.

Example 2: Cloning and sequence analysis of CsGH3.1 gene

1) Extraction of total RNA from tea tree leaves and synthesis of cDNA

Weigh 100 mg of ground tea tree leaves, use SV Total RNA Isolation System (Promega) to extract total RNA and detect its concentration and purity; 1 μg of total RNA is used for cDNA synthesis (PrimeScript ^TM RT-PCR Kit, TaKaRa), and the specific operation refers to the product manual.

2) Cloning of CsGH3.1 gene

HS DNA Polymerase(TaKaRa)说明书。扩增产物经电泳检测后(图2)，利用AxyPrep^TMDNA Gel Extraction Kit(Axygen)进行回收纯化；由于PrimeSTAR HS高保真DNA聚合酶的扩增产物大部分为平滑末端，因此纯化后的片段连入平末端克隆载体Blunt Zero并转入大肠杆菌TG1细胞；挑选阳性克隆保存、测序。引物序列如下：Using the above cDNA as a template, amplify the ORF sequence of the CsGH3.1 gene, refer to the reaction system and procedures

HS DNA Polymerase (TaKaRa) instructions. After the amplified product was detected by electrophoresis (Figure 2), it was recovered and purified using AxyPrep ^TM DNA Gel Extraction Kit (Axygen); since most of the amplified products of PrimeSTAR HS high-fidelity DNA polymerase have blunt ends, the purified fragments were ligated Enter the blunt-end cloning vector Blunt Zero and transform into E. coli TG1 cells; select positive clones for preservation and sequencing. The primer sequences are as follows:

GH3.1-F2: 5'-ggatcttccagagatATGGCGGTTGATTCTGTTTTG-3' (shown in SEQ ID No.7)

GH3.1-R2: 5'-ctgccgttcgacgatTCACCGACGGCGTTCAGG-3' (shown in SEQ ID No. 8).

3) OsHR1 gene sequence analysis

The sequencing result is shown in SEQ ID No.1. According to the open reading frame (ORF) of the sequence, the amino acid sequence of the protein encoded by the gene is deduced, see SEQ ID No.2.

Example 3: Obtaining of CsGH3.1 Gene Overexpression Arabidopsis Strains

1) Construction of CsGH3.1 overexpression vector

Primers GH3.1-F2 and GH3.1-R2 were designed to amplify the ORF fragment of CsGH3.1, and the product was double digested with KpnI and XbaI, inserted into the CaMV35S promoter of the expression vector pCambia2301 (Figure 3), and transformed into Agrobacterium The strains were preserved and sequenced. The primer information is as follows:

GH3.1-F3: 5'-GG GGTACC ATGGCGGTTGATTCTGTTTTG-3' (as shown in SEQ ID No.9)

GH3.1-R3: 5'-GC TCTAGA TCACCGACGGCGTTCAGGAG-3' (shown in SEQ ID No. 10).

2) Transformation of Arabidopsis thaliana by soaking flowers

Select the Arabidopsis thaliana that has just begun to bloom (or remove the top flower buds when bolting to 4-5cm to promote the growth of its side buds, and then when the side buds grow to just flowering), standby; pick Agrobacterium to LB that adds corresponding antibiotics In the culture medium, shake the bacteria, centrifuge to enrich the bacteria, discard the supernatant; resuspend the Agrobacterium in the infiltration solution; pour the infiltration solution into a suitable Erlenmeyer flask, put the Arabidopsis upside down on the Erlenmeyer flask to ensure Submerge the part above the rosette leaves in the liquid, shake gently, and place it for about 10 seconds; take out the plant, shake it for 3 seconds, to remove excess liquid, then place it horizontally in a black plastic bag to maintain humidity, avoid high light and high temperature, place it for 24 hours, and then Continue to cultivate normally; after the seeds mature, collect the T ₀ generation seeds.

3) Screening and identification of overexpressed Arabidopsis homozygous lines

The transgenic seeds of the T ₀ generation were sown in 1/2 MS medium containing kanamycin for resistance selection, the surviving plants were marked and transplanted for further cultivation, and the collected seeds were marked as the T ₁ generation. The seeds of the _T1 generation were continuously screened for resistance, and the collected seeds were marked as the _T2 generation; the seeds of the _T2 generation were continuously screened for resistance, and the germination rate of the seeds was counted. The female parent of the T ₂ generation without trait segregation is homozygous. The homozygous plants were line marked and transplanted. The collected seeds were marked as T ₃ generation, and the seeds of this generation were homozygous; the T ₃ generation seeds were continued to be sown in 1/2 MS medium containing kanamycin for purification and identification. The seedlings were taken to extract RNA for PCR identification and GUS staining ( FIG. 4 ).

The overexpression lines were further identified by specific PCR amplification. The results showed that the specific band of CsGH3.1 could be identified in the overexpression lines, but there was no corresponding characteristic band in the wild type, indicating that CsGH3.1 had been successfully transformed into Arabidopsis thaliana and was successfully expressed at the transcriptional level ( Figure 4a). To further identify whether CsGH3.1 was successfully expressed at the protein level, Arabidopsis seedlings were stained with GUS. Taking the wild type decolorized to colorless as the control, it can be seen that the overexpression lines have obvious GUS activity as a whole, indicating that the CsGH3.1 protein has been successfully expressed in Arabidopsis (Fig. 4b). The above results show that this experiment has successfully obtained the CsGH3.1 overexpression strain, which can be used in subsequent experiments. After homozygous identification, the remaining seedlings were transplanted and cultured until maturity.

Example 4: Study on the Function of CsGH3.1 Transgenic Arabidopsis Lines Regulating Arabidopsis Collateral Differentiation

1) Phenotype analysis of Arabidopsis plants overexpressing CsGH3.1

After 4 weeks of culture under long-day conditions, the growth of CsGH3.1 overexpressed Arabidopsis and wild type began to show differences, and two typical plants CsGH3.1-OX-Line1 were selected from Arabidopsis overexpressed lines , CsGH3.1-OX-Line4 were compared with wild type (Fig. 5a). When cultured to 10 weeks, the phenotypes of overexpressed and wild-type plants showed significant differences (Fig. 5b). Compared with the wild type, the two overexpression lines were significantly dwarfed, accompanied by a significant increase in the number of secondary branches, and the number of primary branches decreased to the base of the stem.

In order to further analyze the specific differences in side branch phenotypes between overexpressed and wild-type plants, the relevant data of overexpressed plants and wild-type primary and secondary branches were counted. It can be seen from Figure 6 that the number of secondary branches of the overexpressed plants increased, the length shortened, and the tillering site decreased, while the number and length of the primary branches were not significantly different from those of the wild type. The above data indicated that the effect of CsGH3.1 overexpression on Arabidopsis collateral growth was mainly reflected in the development of secondary branches.

2) Tissue-specific expression analysis

In order to observe the expression activity of CsGH3.1 overexpression vector in Arabidopsis tissues, the relative gene expression of CsGH3.1 in different tissues of overexpressed plants was determined. CsGH3.1 was significantly expressed in the leaves, flowers, and fruit pods of the overexpressed plants, and the expression level was the highest in leaves, followed by fruit pods (Fig. 7a).

Considering that CsGH3.1 is a subfamily II gene, it mainly catalyzes the combination of free IAA and amino acids to form IAA amide conjugates to maintain auxin homeostasis, and several subfamily II genes have been confirmed to have amide catalytic enzyme activity in other species. . In this study, the expression levels of 8 AtGH3 subfamily II genes were measured, and the wild type was used as the control. The results showed that AtGH3.1, AtGH3.4, AtGH3.6, AtGH3.9 and AtGH3.17 were all significantly induced by the overexpression of CsGH3.1, especially the expression of AtGH3.4 was about 58 times that of the wild type (Fig. 7b). The above results indicated that inducing the expression of Arabidopsis AtGH3 subfamily II members may be one of the mechanisms by which CsGH3.1 overexpressed Arabidopsis plants regulate the homeostasis of endogenous auxin.

sequence listing

<110> Tea Research Institute of Chinese Academy of Agricultural Sciences

<120> A gene related to the regulation of lateral branch differentiation of tea tree origin, its encoded product and its application

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<170> SIPOSequenceListing 1.0

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Thr Leu Asp Tyr Tyr Ser Gly Arg Leu Pro Leu Ala Cys Thr Met Tyr

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Ala Ser Ser Glu Cys Tyr Phe Gly Leu Asn Leu Asn Pro Met Ser Lys

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Phe Leu Pro His Glu Pro Asn Ser Ala Glu Ser Thr Arg Tyr Ser Pro

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Leu Leu Val Thr Thr Tyr Ala Gly Leu Cys Arg Tyr Arg Val Gly Asp

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Glu Phe Asn Thr Ser Val Val Glu Tyr Thr Ser Tyr Ala Asp Thr Lys

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gctctagatc accgacggcg ttcaggag 28

Claims (1)

1. The application of a gene CsGH3.1 related to the regulation of lateral branch differentiation of tea tree origin in increasing the number of secondary branches of plants, reducing the tiller node position and reducing the length of secondary branches, the DNA sequence of the gene is as SEQ ID No. 1.

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